Previous research led to the discovery that HIV-1 utilizes TSG101/ESCRT-I and the cellular pathway of MVB vesicle formation to bud from cells. Our studies also provided a general overview of the individual components of the MVB pathway, their pairwise interactions, and three dimensional structures of a series of MVB proteins and complexes, including: the TSG101 UEV/PTAP complex, the ALIX YPXL binding domain, VPS4 MIT and ATPase domains, and ubiquitin complexes with the TSG101 UEV, Npl4 NZF, and EAP45 GLUE domains. Nevertheless, we do not yet understand the basic mechanisms that underlie most essential pathway functions including regulation, protein sorting, membrane deformation, and membrane fission. We now propose biochemical, mechanistic, and structural studies of three different MVB complexes that function in HIV-1 budding. Studies in Aim 1 will focus on the biochemistry of human ESGRT-I. ESCRT-I binds directly to the PTAP late domain of HIV-1 Gag and facilitates virus release. We have recently developed systems for expressing and purifying recombinant ESCRT-I complexes and discovered a fourth family of previously uncharacterized ESCRT-I subunits. We will now build on these observations to define the precise composition of ESCRT-I, test models for ESCRT-I activation and regulation, and characterize a series of ESCRT-I binding proteins. Studies in Aim 2 will focus on the structure and function of the ALIX/AIP1 protein, which binds directly to YPXL late domains and faciliates virus budding. We have developed systems for analyzing the role of ALIX in HIV-1 release and determined the crystal structure of the central YPXL binding region of ALIX. We will now map the YPXL binding site, test the hypothesis that late domains induce conformational changes that activate ALIX, and characerize the roles of ALIX binding partners in virus budding. Studies in Aim 3 will focus on understanding how the VPS4 ATPases interact with the ESCRT-III lattice and release the assembled MVB machinery from the membrane. VPS4A and VPS4B are the only known enzymes in the MVB pathway, and their ATPase activity is required for release of HIV-1 and many other viruses. We now propose to determine how VPS4 recognizes ESCRT-III substrates and to test the model that VPS4 functions as a specialized chaperone that uses the energy of ATP hydrolysis to unfold ESCRT-III subunits, release them from the membrane, and possibly also drive vesicle/virus release.